Surgical applications for bmp binding protein

ABSTRACT

The present invention relates to the clinical application of BBP, alone or in combination with other growth factors, for use in bone healing applications, such as spinal surgery. Additional applications include use in orthopedic implantable prostheses and implantation into other surgical sites (e.g., surgical reconstruction, regional osteopenia, etc.) where bone is desired.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.12/448,497, filed Jun. 22, 2009, which is a national stage applicationof PCT International Patent Application No. PCT/US2007/026315, filedDec. 26, 2007, which claims priority to U.S. Provisional PatentApplication No. 60/876,821, filed Dec. 22, 2006.

This application is a continuation of U.S. patent application Ser. No.12/448,497, filed Jun. 22, 2009, which is a continuation-in-part of U.S.patent application Ser. No. 11/985,745, filed Nov. 16, 2007, which is acontinuation-in-part of U.S. patent application Ser. No. 10/587,313,which has a 371 date of Apr. 28, 2008, and which is a national stageapplication of PCT International Patent Application No.PCT/US2005/002722, filed Jan. 28, 2005, which claims priority to U.S.Provisional Patent Application No. 60/539,903, filed Jan. 28, 2004.

BACKGROUND OF THE INVENTION

Growth factors are substances, including peptides, which affect thegrowth and differentiation of defined cell populations in vivo or invitro. Normal bone formation occurs during development, bone remodelingoccurs in adult life, and bone repair occurs in order to preserve theintegrity of the skeleton. Bone formation, remodeling and repair involvebone resorption by osteoclasts and bone formation by osteoblasts. Celldifferentiation and the activity of osteoblasts and osteoclasts areregulated by growth factors. Thus, any interference between the balancein cell differentiation and resorption can affect bone homeostasis, boneformation and repair.

Osteoblasts are derived from a pool of marrow stromal cells (also knownas mesenchymal stem cells). MSC are present in a variety of tissues andare prevalent in bone marrow stroma. MSC are pluripotent and candifferentiate into chondrogenic or osteogenic cells includingosteoblasts, chondrocytes, fibroblasts, myocytes, and adipocytes.

The induction of ectopic bone formation by demineralized bone matrix(DBM) has been described. (Urist, M. R.: Bone: Formation byautoinduction. Science 150:893-899, 1965; Urist, et al., Purification ofbovine morphogenetic protein by hydroxyapatite chromatography. Proc.Natl. Acad. Sci. USA 81:371-375, 1984; Urist, M. R. Emerging concepts ofbone morphogenetic protein. In Fundamentals of Bone Growth: Methodologyand Applications, Boston C.R.C. Press, pp. 189-198, 1991.) Further, theproperties of the partially purified protein fraction, bone morphogenicprotein/non-collagenous protein (“BMP/NCP” or “BMP”s) have beendescribed. (Urist, et al. Methods of Preparation and Bioassay of BoneMorphogenetic Protein and Polypeptide Fragments. In Methods inEnzymology. Vol. 146. New York, Academic Press, pp. 294-312, 1987;Urist, et al., Hydroxyapatite affinity, electroelution, andradioimmunoassay for identification of human and bovine bonemorphogenetic proteins and polypeptides. In Development and Diseases ofCartilage and Bone Matrix. New York, Alan R, Liss, Inc., pp. 149-176,1987.)

BMP/NCP was never purified to homogeneity, but other investigators haveused similar starting materials to clone a number of recombinant “BMPs.”However several of these molecules have little or no osteogenicactivity. “BMPs” and other osteogenic factors have been studied for usein clinical applications. However, the cost of using minimally effectivedosages of BMP-7 (also known as OP-1), for example has been a limitingfactor in clinical use. Therefore, effective and affordable compositionsand methods are desired for clinical applications relating to bone.

Adjuvant therapeutics to enhance bone healing are important in manyaspects of orthopedics, but are especially important for spinal fusionwhere prompt and thorough osteogenesis is critical. The most usefulagents currently available include bone growth factors, such as bonemorphogenetic proteins (BMPs). These proteins induce the recapitulationof endochondral bone formation among undifferentiated mesodermal cells.However, their usefulness is limited by expense and by local adverseeffects such as unwanted ectopic bone formation and inflammatoryresponses associated with doses currently used for spinal fusionprocedures.

Furthermore, potential systemic adverse effects of high dose BMPs whento be used for longer fusions has not been fully determined Therefore, anumber of strategies are being developed to provide safer, lessexpensive and more efficacious adjuvant agents.

The use of BMP-2 as a bone generator for spinal fusion is gainingincreased popularity. Recent studies have shown that it may be usedeffectively for both anterior and posterior fusion procedures throughthe whole spinal column. However, its high expense as well as thereported local side effects such as unwanted bone formation anddangerous swelling at the neck after the anterior cervical fusionprocedure has prevented its extensive use for spinal procedures. Focushas now shifted to increase the effectiveness of BMP-2 while decreasingits dose and controlling the side effects. The main limitation is theneed for better delivery systems that provide a sustained, biologicallyappropriate concentration of BMP-2 at the side of fusion bed. Deliveryneeds to be sustained, because BMPs have exceedingly short biologicalhalf-lives, usually the order of minutes or hours, rather than the daysor weeks needed to stimulate a complete osteogenic response. Forexample, rBMP-2 has been described as having a half-life of only a fewhours.

BRIEF SUMMARY OF INVENTION

The inventions are related to a cyclic peptide designated BMP BindingPeptide (BBP) that avidly binds growth factors, such as rhBMP-2. BBPincreases the rate and degree to which rhBMP-2 induces bone formation.BBP may accomplish this by increasing the residency rates of other bonegrowth factors. However, BBP alone induces calcification ofchondrogenic, osteogenic and osteoblastic cells. Compositions andsubstrates including BBP, antibodies to BBP and methods of using BBP areuseful in applications relating to bone.

In one embodiment, the invention may include a method of treatment withagents for maintaining bone homeostasis, enhancing bone formation and/orenhancing bone repair.

In one embodiment, the invention includes methods and devices forincreasing the residency times of bone inducing substances, such as BMP.In one embodiment, the invention includes methods and devices forincreasing the rate and overall osteogenic activity of bone inducingsubstances, such as BMP. Further, in one embodiment, the inventionincludes methods and devices for reducing time required for the effectof osteogenesis and calcification of bone inducing substances.

In one application of the invention, the method may be applied to inducethe local repair of bone or to treat bone related disorders, such asosteoporosis. In one embodiment of the invention, BBP may be used toinduce the effects of other bone growth factors on bone fusion inmembraneous bone (such as spinal fusion) or in endochondral bone.

In one embodiment, the invention may include implants having agents orseeded with pluripotential or differentiated cells for inducing boneformation or repair. The invention may also include the application ofsubstances or differentiated cells at a site where bone formation orbone repair is desired.

This invention is advantageous at least in that BBP alone or incombination with other growth factors enhances calcification ofchondrogenic or osteogenic precursor cells. Further, this invention isadvantageous at least in that BBP enhances osteogenesis to occur fasterto a greater extent, which may improve the clinical rate andeffectiveness of treatment with BMP, and reduce doses and therefore thecosts and side effects of BMP treatment alone.

These, as well as other objects, features and benefits will now becomeclear from a review of the following detailed description ofillustrative embodiments and the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A are BBP bovine (1) amino acid and (2) nucleic acid sequences,respectively; FIG. 1B is a partial amino acid sequence of the bovine BMPbinding protein (“BBP”) showing the cystatin homology region, the BMP-2homology region, and the TGF-β receptor II homology domain.

FIG. 2 is an amino acid sequence alignment of human BMP-2 and the BMP-2homology region in bovine SPP-24; (i, identical; c, conservativesubstitution; sc, semi-conservative substitution).

FIG. 3 is an amino acid sequence alignment of bovine fetuin and humanTGF-β receptor II (above) and of human TGF-β receptor II and the TGF-βreceptor II homology domain of bovine SPP-24 (corresponding to BBP)(bottom); (i, identical; c, conservative substitution; sc,semi-conservative substitution).

FIG. 4 is a radiogram of mouse hindquarters 21 days after implantationof 500 μg of BBP in atelocollagen (top) or atelocollagen alone (bottom).

FIG. 5 is a histological section of mouse muscle 21 days afterimplantation of 500 μg of BBP in atelocollagen. (H & E stain. Originalmagnification 100×.)

FIG. 6 are radiograms of mouse hindquarters 21 days after implantationof 5 μg of rhBMP-2 (left) or 5 μg of rhBMP-2 plus 500 mg of BBP (right).

FIG. 7 are radiograms of mouse hindquarters 9 (top) and 12 (bottom) daysafter implantation of 5 μg of rhBMP-2 (left) or 5 μg of rhBMP-2 plus 500mg of BBP (right).

FIGS. 8A-B are histological sections of mouse hindquarters 9 days afterimplantation of 5 μg of rhBMP-2 alone (8A) or 5 μg of rhBMP-2 plus 500μg of BBP (8B).

FIG. 9 is a surface plasmon resonance sensogram for the interaction ofrhBMP-2 (affixed to the chip) and cyclized BBP at concentrations rangingfrom 1×10⁻⁵ M 1×10⁻⁴ M.

FIG. 10 is a bar graph depicting the percentage of rhBMP-2 retentionover 1, 3 and 7 days in the presence or absence of BBP.

FIG. 11 includes amino acid sequences against which specific SSP-24/BBPantibodies have been generated.

FIGS. 12A-B depict flowcharts of exemplary methods of the invention.

FIGS. 13A-B are schematic depictions of two embodiments of theinvention.

FIG. 14A is a chart showing the amino acid sequences for BPP in variousspecies (SEQ ID NOS: 11, 1 and 12-19, respectively, in order ofappearance). FIG. 14B is a list of the nucleic acid sequences for BPP invarious species (SEQ ID NOS: 20-28, respectively, in order ofappearance).

FIG. 15 is an anterior-posterior radiograph of a rat spine fused atL4-L5 with the application of BBP high dose (1000 μg)+rhBMP-2 low dose(1 μg) 8 weeks after treatment.

FIG. 16 is an anterior-posterior radiograph of a rat spine showingpseudoarthritis at right anno fusion at left L4-L5 with the applicationof rhBMP-2 (1 μg) treatment.

FIG. 17 is a histological section of rat spinal region 8 weeks aftertreatment of a combination of BBP and rhBMP-2. (H & E stain. Originalmagnification 8.4×.)

FIG. 18 is a histological section of rat spinal region 8 weeks aftertreatment with low dose rhBMP-2 (1 μg). (H & E stain. Originalmagnification 8.4×.)

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

One embodiment of the invention comprises a peptide having the aminoacid sequence of SEQ ID NO: 1. The bovine derived amino acid SEQ ID NO:1 has been designated BBP, and SEQ ID NO: 2 corresponds to the bovinenucleic acid sequence encoding BBP.

One embodiment of the invention comprises a peptide having the aminoacid sequence of SEQ ID NO: 12, which is the sequence of human BBP. SEQID NO: 21 corresponds to the human nucleic acid sequence encoding humanBBP.

BBP is a 19 amino acid, 2.1 kD peptide, derived from a 18.5 kD fragmentof a known 24 kD secreted phosphoprotein (“SPP-24”). SPP-24 isillustrated by SEQ ID NO: 2. Notably, SPP-24 inhibits BMP-2 induced boneformation. BBP contains the cystatin-like domain of SPP-24. BBP isexpressed at least in the liver and bone (including at leastdemineralized cortical bone and periosteum).

The BBP amino acid sequence is similar to the TGF-β/BMP-binding regionof fetuin, a member of the cystatin family of protease inhibitors. BBPavidly binds rhBMP-2 (recombinant human BMP-2) with a K_(D) of ×10⁻⁵ M.BBP may also bind other molecules having similar binding domains toBMP-2, such as other TGF-β proteins (including but not limited to BMP-4,BMP-7 (OP-1), BMP-6, BMP-8 (OP-2), BMP-9 and TGF-β) and affect theirretention rates and/or activity as well. There is a high degree ofsequence similarity (about 90%) between BMP-4 and BMP-2, for example.Additionally, the various ostroinductive BMPs (BMP-2, BMP-4, BMP-6,BMP-7 and BMP-9) have been demonstrated to bind to the same receptors(with some variation in affinities). Therefore, given the sequence andbinding similarities, binding with these additional BMPs is expected.Additional growth factors useful in this invention may include: GDF5,and other BMPs such as, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9.

BBP alone induces calcification of vertebrate chondrogenic andosteogenic precursor cells. BBP increases the increases the rate anddegree to which rhBMP-2 induces bone formation. Surprisingly, BBPcombined with BMP-2 in vivo causes osteogenesis to occur faster and to agreater extent and with smaller amounts of rhBMP-2, than compared withthe effect of BMP-2 alone. This result was unexpected given the role ofSSP-2 which inhibits bone formation by BMP-2, as discussed above.

For example, when implanted alone in mouse muscle, the BBP inducesdystrophic calcification. The process of bone formation in repair orectopic bone formation the “mouse hindquarter” or “muscle pouch” modelrecapitulates endochondral bone formation. The first step involves theproduction of cartilage, which is replaced by bone. This same processthat occurs during endochondral bone formation in development, whilesome membraneous bone formation occurs directly without a cartilageintermediary.

In one embodiment of the invention, a peptide comprising a fragment ofBBP may be useful, if the fragment similarly increases degree or rate ofosteogenesis by BMP-2 in mammalian cells, or increases degree or rate ofcalcification in vertebrate cells, or specifically mammalianchondrogenic or osteogenic progenitor cells.

Forms of BBP having modifications of the amino acid SEQ. ID NO: 1 mayalso be useful in this invention. For example, the conserved amino acidsequences of BBP between species, deletional or insertionalmodifications, conservative or semi-conservative substitutionalmodifications are intended to be encompassed in the claimed BBP, to theextent that the modified amino acid sequences increase the residencytime and or activity of BMP-2 or other TGF-β homologous molecules. BBPis a β-pleated sheet-turn-sheet pleated sheet molecular motif (“B-T-B”).It is currently believed that growth factor binding amino acids residein the T-section. Therefore, amino acid substitutions in the T-sectionmay affect activity of BBP to a greater extent than substitutions in theB regions.

As above, BPP is believed to increase the effect of growth factors, likeBMP-2 by increasing the amount of time (“residency time”) that a growthfactor remains at the site of implantation, and/or the overall activityof BMP-2 or other TGF-β homologous molecules. BMP alone, for example, israpidly removed from implantation sites, while implantation of BMP withBBP has been shown to increase residence time. The increase in residencytime may be attributed to, for example: 1) BPP's ability to increase thehalf-life of growth factors (such as by reducing the rate ofproteolysis), and/or 2) decrease the diffusion rate of growth factorsfrom an application site.

One embodiment of the invention comprises a peptide having the sequenceof SEQ ID NO: 11: C-R-S-T-V-X-Z-S-X-X-X-V-X-X-V X-Z-Z-C, which is themammalian consensus sequence for BBP. FIG. 14A shows the homology inamino acid sequence across bovine (SEQ ID NO: 1; nucleic acid sequenceset forth at SEQ ID NO: 2), human (SEQ ID NO: 12; nucleic acid sequenceset forth at SEQ ID NO: 21 (position 9 is either A or V), porcine (SEQID NO: 13; nucleic acid sequence set forth at SEQ ID NO: 22), ovine (SEQID NO: 14; nucleic acid sequence set forth at SEQ ID NO: 23), rat (SEQID NO: 15; nucleic acid sequence set forth at SEQ ID NO: 24), and mouse(SEQ ID NO: 16; nucleic acid sequence set forth at SEQ ID NO: 25) BBP.FIG. 14A also shows highly conserved regions in chicken (SEQ ID NO: 17;nucleic acid sequence set forth at SEQ ID NO: 26), salmon (SEQ ID NO:18; nucleic acid sequence set forth at SEQ ID NO: 27) and trout (SEQ IDNO: 19; nucleic acid sequence set forth at SEQ ID NO: 28).

In FIG. 14A, “X” and “Z” are used to denote amino acid substitutionsthat are understood to be semi-conservative or conservative,respectively. Conservative substitutions include amino acids selectedfrom the same group, and semi-conservative substitutions includesubstitutions that are not believed to affect the BMP-2 binding domainor the function of the BBP. For example, the substitution at position 6is conservative between human, rat and ovine, but semi-conservative withsome other species because the amino acids reported at that position indifferent species are: Q and E (Q in porcine, rat, and mouse BBP, and Ein chicken). Although K and R are both classified as basic amino acids,Q is classified as an uncharged polar amino acid, therefore thesubstitution is not conservative. The substitution is semi-conservative,however, because the function of BBP is believed to be unaffected.Semi-conservative substitutions are also found at positions 9, 10, 11,and 16. At position 9, the amino acids A is found in bovine, human,porcine and ovine BBP, compared to K in rat and mouse BBP. At position10, the amino acid E is reported for bovine, porcine, and ovine BBP,human BBP contains Q at that position, and rat and mouse BBP contain theamino acid G. At position 11, the amino acid Q is found in bovine,human, rat and mouse, whereas K, is reported for porcine BBP and R forovine BBP. At position 16, W is found in bovine, porcine, ovine, rat andmouse BBP, whereas human BBP contains an H. There are alsosemi-conservative substitutions at positions 13 and 14 betweenrat/human, as opposed to other species.

An example of a conservative substation is found at position 7. At thisposition, different hydrophobic amino acids are observed in differentspecies, namely, M in bovine, ovine, rat, and mouse BBP, compared to Vin human and I in porcine BBP. This substitution is consideredconservative because M, V, and I are all hydrophobic amino acids. Otherconservative substitutions occur at positions 17 and 18. Two hydrophobicamino acids, A and V, are found at position 17. At position 18, twobasic amino acids, R and H, are found.

One embodiment of the invention may be a composition including BBP whichincreases degree or rate of calcification in vertebrate cells, or morespecifically mammalian chondrogenic or osteogenic precursor cells.Further, the invention may be including BBP which increases degree orrate of osteogenesis by BMP-2, and one of BMP-2 or demineralized bonematrix. Further, the composition may additionally or alternativelyinclude other TGF-β family members, including but not limited to BMP-4or BMP-7 separately or mixtures thereof.

In one embodiment, the invention may include a medicament for use ininducing the rate or degree of osteogenesis in a vertebrate including atherapeutically effective dosage of BBP alone or in combination with agrowth factor, such as BMP or DBM. The invention may further include, amedicament for use in inducing the rate or degree of calcification in avertebrate including a peptide comprising BBP.

In one embodiment, BBP or the combination of BPP and one or more othergrowth factors may be placed on or in a substrate or carrier, such as acollagen sponge or orthopedic implant or prosthesis, for implantation ata site where bone formation and/or implant integration is desired.

Applications for BBP. A number of applications for BBP are suggestedfrom its pharmacological (biological activity) properties. For example,BBP alone or in combination with other TGF-family members such as BMP-2,BMP-4 and BMP-7, or demineralized bone matrix may be used in clinical orresearch methods for inducing bone formation, maintaining bonehomeostasis and/or enhancing bone repair. BBP may be used alone or incombination to treat developmental or homeostatic bone disorders (suchas osteoporosis), bone injury (such as fracture healing flat (e.g.,membranous) and long (e.g., endochondral) bones, non-union fractures andreconstructive surgery. The invention may also be used in treatingperiodontitis, periodontal regeneration, alveolar ridge augmentation fortooth implant reconstruction, treatment of non-union fractures, sites ofknee/hip/joint repair or replacement surgery.

By way of example, BMP has been demonstrated to be effective inproviding spinal fusion, but has limitations to widespread use due toadverse side effects, such as causing inflammation and ectopic boneformation, particularly in the cervical spine. In one embodiment BBP maybe used in conjunction with lower doses of BMP to preserve its efficacy,an increase its retention at the application site. As discussed herein,BBP has been demonstrated to bind BMP, including recombinant human BMP-2enabling a two fold retention of rhBMP-2 for up to seven days in vitroand in vivo.

Clinical indices of a method or compounds ability to maintain bonehomeostasis is evidenced by improvements in bone density at differentsites through out the body as assessed, at least by DEXA scanningEnhanced bone formation in a healing fracture is routinely assessed byregular X-ray of the fracture site at selected time intervals. Moreadvanced techniques for determining the above indices, such asquantitative CT scanning or quantitative histological methods (e.g.,tissue is processed, stained, and microscopically examined and bonedefined an measured with image analysis) may be used. Further, measuresof bone density, bone area, bone mineral content, formation of ectopicbone, and increases in the opacity of tissue upon X-ray examination,expression of alkaline phosphatase activity, calcium incorporation,mineralization or expression of osteocalcin mRNA may be used to observethe effects of BBP calcification and/or osteogenesis

The invention may also include the use of agents which inhibitosteoclastic bone resorption. Agents which may be useful in thisinvention to effect osteoclastic bone resorption include, but are notlimited to, bisphosphonates, the selective estrogen receptor modulators,calcitonin, and vitamin D/calcium supplementation. The invention mayalso include the use of agents which induce osteoblastic bone formation.Agents which may be useful in this invention include, but are notlimited to PTH, sodium fluoride and growth factors, such as insulin-likegrowth factors I and II.

The in vivo models used to show the calcification effects of BBP aloneor osteogenic effects in combination with BMP have been used previouslyin demonstrating similar behaviors of other compounds. In particular, invivo models have also previously been able to successfully predict thein vivo osteogenic effects of compounds such as BMP and insulin likegrowth factors (IGF). Specifically, it has been demonstrated that theosteogenic effects of BBP in an animal model using rat femur, ectopicbone formation model. Therefore it is anticipated that, based on thesesimilar findings, BBP will have osteogenic effects in vivo in humans.Demonstration of osteogenic effects of a compound in these in vivomodels are necessary prior to trials that would demonstrate theireffects in vivo humans.

Therapeutically effective dose. A therapeutically effective dose of BBPor a TGF-β family member useful in this invention is one which has apositive clinical effect on a patient or desired effect in cells asmeasured by the ability of the agent to enhance calcification orosteogenesis, as described above. The therapeutically effective dose ofeach agent can be modulated to achieve the desired clinical effect,while minimizing negative side effects. The dosage of the agent may beselected for an individual patient depending upon the route ofadministration, severity of the disease, age and weight of the patient,other medications the patient is taking and other factors normallyconsidered by an attending physician, when determining an individualregimen and dose level appropriate for a particular patient.

This invention is advantageous and unexpected us in at least the dosageof BMP-2 required to induce a given rate or degree of osteogenesis maybe reduced when BMP-2 is combined with BBP. This is advantageous atleast in reducing the cost of treatment, as BMP can be costly for someapplications. Further, reducing treatment levels of BMP (or other bonegrowth factors) by treating in combination with BMP may also reduce sideeffects which are related to the amount of BMP used, including forexample, inflammation within the soft tissue of the neck or ectopic boneformation. Thus, the use of BBP as a bone growth factor binding agent toincrease exogenous growth factor retention, can aid in overcomingnegative aspects of growth factor use by reducing the amount of growthfactor needed to achieve healing.

Dosage Form. The therapeutically effective dose of an agent included inthe dosage form may be selected by considering the type of agentselected and the route of administration. The dosage form may include aagent in combination with other inert ingredients, including adjutantsand pharmaceutically acceptable carriers for the facilitation of dosageto the patient, as is known to those skilled in the pharmaceutical arts.

Therapeutic formulations of BBP (when claimed is intended to includemodifications or fragments thereof), may be prepared for storage bymixing the BBP having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers, in theform of lyophilized cake or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; anti-oxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins. Other components caninclude glycine, blutamine, asparagine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, Pluronics orpoly(ethylene glycol) (PEG).

The dosage form may be provided in preparations for subcutaneous (suchas in a slow-release capsule), intravenous, intraparitoneal,intramuscular, peri- or intraskeletal for example. Any one or acombination of agents may be included in a dosage form. Alternatively, acombination of agents may be administered to a patient in separatedosage forms. A combination of agents may be administered concurrent intime such that the patient is exposed to at least two agents fortreatment.

Additional Agents. The invention may include treatment with anadditional agent which acts independently or synergistically with BBP toenhance calcification osteogenesis. For example, BBP may be combinedwith BMP, bisphosphonates, hormone therapy treatments, such as estrogenreceptor modulators, calcitonin, and vitamin D/calcium supplementation,PTH (such as Forteo or teriparatide, Eli Lilly), sodium fluoride andgrowth factors that have a positive effect on bone, such as insulin-likegrowth factors I and II and TGF-β. Those skilled in the art would beable to determine the accepted dosages for each of the therapies usingstandard therapeutic dosage parameters, or reduced dosages where theeffects of BBP are synergistic with the secondary agent, such as BMPs.

BBP is currently thought to act upon BMP-2 at least by increasing itsresidency time with a substrate. One embodiment of the invention is amethod of detecting the ability of BBP to enhance the residency time ofa TGF-β homologous molecule including applying an amount of the TGF-βhomologous molecule at a first and second selected location. Further,applying a selected amount of BBP at the first selected location, andfinally detecting the amount of the TGF-β homologous molecule at thefirst and second location after a selected time period; and calculatingthe difference between the amount of the TGF-β homologous molecule atthe first and second location.

In one embodiment, the invention may include a method of enhancing therate or degree of osteogenesis in vertebrate tissue includingapplication of BBP which increases degree or rate of osteogenesis byBMP-2 in mammalian cells and one of a TGF-β family member, such as BMP-2or demineralized bone matrix.

In one embodiment, the invention may include a method of inducingcalcification of vertebrate tissue, or more specifically vertebratechondrogenic or osteogenic precursor cells, including application ofBBP.

In one embodiment, the invention may include a method of enhancing therate or degree of osteogenesis in vertebrate tissue includingadministering chondrogenic or osteogenic precursor cells to the patientat a location proximate to the desired location of osteogenesis;further, administering BBP, and administering one of a TGF-β familymember, such as BMP-2 or demineralized bone matrix.

In one embodiment, the invention may include a method of enhancing therate or degree of calcification in vertebrate tissue includingadministering osteogenic cells to the patient at a location proximate tothe desired location of calcification and further, administering BBP.

In one embodiment, the invention may include method of enhancing therate or degree of osteogenesis in a vertebrate including treatingvertebrate undifferentiated mesynchymal stem cells with one of a TGF-βfamily member, such as BMP-2 or demineralized bone matrix to induceosteogenesis of the cells. Further, treating the vertebrate mesynchymalstem cells with BBP; and administering the vertebrate mesynchymal stemcells to the patient at a location proximate to the desired location ofosteogenesis.

For example, mammalian cells, such as mesenchymal stem cells can beharvested, from the patient or a cell donor. The cells may be injectedin a location where bone formation or repair is desired (such as afracture site or implant site where bone growth is needed), or firsttreated with BBP and/or BMP. The cells may then be re-administered tothe patient, either systemically or at a selected site at whichosteogenesis of calcification is desired. Additionally, the patient mayby treated locally or systemically with at least one additional agentwhich effects osteogenesis or calcification.

FIGS. 12A-B depict flowcharts of exemplary methods of the invention, thesteps of which may be performed in any order.

One embodiment of the invention may include an article of manufacturecomprising BBP immobilized on a solid support. The solid support mayfurther include a TGF-β family member, such as BMP-2 or demineralizedbone matrix.

One embodiment of the invention may include an implant for use in vivoincluding, a substrate where at least the surface of the implantincludes BBP. The implant may further include MSC, chondrocytic orosteoblastic progenitor cells. Further, the implant may be formed intothe shape of a pin, screw, plate, or prosthetic joint, for example.

For example, FIGS. 13A-B depict two embodiments of the presentinvention. In FIG. 13A, the invention may include implants or grafts(200) for use in the body comprising, a substrate having a surface(201), wherein at least the surface of the implant includes BBP (203) inan amount sufficient to induce, calcification or osteogenesis in thesurrounding tissue. The implant may include mesynchymal stem cell,chondrogenic or osteogenic cells expressing BBP, and/or BMP-2,demineralized bone matrix, or collagen cultures. The implant may be inthe form of, but are not limited to pins, screws, plates or prostheticjoints which may be placed in the proximity of or in contact with a bone(202) that are used to immobilize a fracture, enhance bone formation, orstabilize a prosthetic implant by stimulating formation or repair of asite of bone removal, fracture or other bone injury (204).

As shown in FIG. 13B, the invention may also include the in vitro (suchas on cultures of collagen or chondrocytes) or in vivo application of ata least BBP containing composition or BBP expressing cells (206) in theproximity of or in contact with a bone (202), an implant (200) at a siteof bone removal, fracture or other bone injury (204) where osteogenesisand/or calcification is desired. The BBP composition may be applied incombination with other agents such as BMP-2, demineralized bone matrix,or collagen cultures.

For example, the use of stem cells for treating bone related disordersin humans has also been examined. Infusion of osteoblastic progenitorstem cells from a healthy individual into a diseased individual has beenshown to improve bone density in these patients. Cells may be pretreatedwith BMP and BPP, or applied concurrently therewith.

In one embodiment, the invention may include a monoclonal or polyclonalantibody having selective binding to any portion of BBP, or the BBPportion of the BBP precursor, SSP-24.

BBP or fragments thereof may be fused (for example by recombinantexpression or in vitro covalent methods) to an immunogenic polypeptideand this, in turn, may be used to immunize an animal in order to raiseantibodies against BBP. Antibodies are recoverable from the serum ofimmunized animals. Alternatively, monoclonal antibodies may be preparedfrom cells from the immunized animal in conventional fashion Immobilizedantibodies may be useful particularly in the detection or purificationof BBP.

Two examples of specific peptide sequences against which rabbitpolyclonal antibodies have been generated include: (1) An antibodyagainst the peptide sequence “IQETTCRRESEADPATCDFQRGYHVPVAVCRSTVRMSAEQV”(FIG. 11-SEQ. ID NO: 3) that reacts with both bovine and human SSP-24,the BBP precursor. This antibody was generated in rabbits immunized withthe synthetic peptide indicated above. Further, (2) An antibody directedagainst the sequence “CGEPLYEPSREMRRN” (FIG. 11-SEQ. ID NO: 4) that wasalso produced in rabbits immunized with a synthetic peptidecorresponding to the indicated sequence. This second antibody reactswith bovine SSP-24. The N-terminal cysteine is not a part of the nativeSSP-24 sequence; but is preferably included to allow the peptide to beconjugated to chromatographic resins for affinity chromatography.Additional peptide sequences may be identified for specific binding toBBP, and sequences may be selected so as to create an antibody havingselective binding with BBP, but so as to not interfere with BBP binding,such as the region of BBP which binds with BMP-2 (including SEQ ID NOS:3 and 4) or other TGF-β family members. These specific peptide sequencescan be used to generate monoclonal antibodies, which preparation methodsare known in the art.

Antibodies against the sequences above, corresponding sequences in themouse, human, and rat genome, or any derivatives of the immunogenicsequences are also useful in this invention. These antibodies are usefulin at least to the extent that they recognize the BBP amino acidsequence with high specificity. Such antibodies may also be useful ininhibiting protein specific interactions of BBP with other moleculeswhere the antibody binds to a location on the peptide which interactswith other molecules. The inhibition of BBP activity in situations wherethe rate or degree of chondogenesis or osteogenesis may be modified.

In one embodiment the invention, antibodies specific for BBP may beuseful in decreasing the degree or rate of osteogenesis by BMP-2 invertebrate cells or decreasing degree or rate of calcification invertebrate cells, or more specifically in mammalian chondrogenic orosteoblastic precursor cells.

One embodiment of the invention may also include a method of using BBPselective antibodies to detect the presence of SSP-24/BBP in sample(including but not limited to a cell culture, tissue sample, peptidefraction, Western blot) including exposing the sample to the BBPselective antibody and visualizing the complex of SSP-24/BBP and BBPantibody.

In one embodiment of the invention, BBP antibodies may be used for theaffinity purification of the BBP from recombinant cell culture ornatural sources. BBP antibodies that do not detectably cross-react withother growth factors can be used to purify BBP from these other familymembers.

In one embodiment, the invention may include a nucleic acid constructcomprising a DNA or RNA nucleic acid sequence encoding BBP, or modifiedsequences corresponding to the modified amino sequences described above.

The invention may also include, an expression vector operatively linkedto a nucleic acid sequence encoding BBP, or precursor SSP-24 Further, atransformant may be obtained by introducing the nucleic acid constructencoding for BBP, or its precursor SSP-24 into a host cell.

Practice of this invention may include the use of an oligonucleotideconstruct comprising a sequence coding for BBP and for a promotersequence operatively linked in a mammalian or a viral expression vector.Expression and cloning vectors contain a nucleotide sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomes, and includesorigins of replication or autonomously replicating sequences. Suchcloning vectors are well known to those of skill in the art. Expressionvectors, unlike cloning vectors, may contain an inducible orconstitutive promoter which is recognized by the host organism and isoperably linked to the BBP nucleic acid. The nucleic acid may beoperably linked when it is placed into a functional relationship withanother nucleic acid sequence. For example, DNA for a pre-sequence orsecretory leader is operably linked to DNA for a polypeptide if it isexpressed as a pre-protein which participates in the secretion of thepolypeptide.

One embodiment of the invention may also include a method of using DNAor RNA nucleic acid sequences complimentary and having specific bindingfor the DNA or RNA sequences encoding BBP to detect the presence of BBPDNA or RNA in a sample, respectively (including but not limited to acell culture, tissue sample, nucleic acid fraction, or Southern orNorthern blot) including exposing the sample to the complimentary BBPDNA or RNA sequences and visualizing the complex of hybrids.

EXAMPLE 1 Extraction and Separation of Non-Collagenous Bone Proteins(NPCs)

Methods: NCPs were extracted from defatted, demineralized human corticalbone powder with 4 M GuHCl, 0.5 M CaCl₂, 2 mM N-ethylmalemide, 0.1 mMbenzamidine HCl, and 2 mM NaN₃ for 18 hr at 6° C. Residual collagen andcitrate-soluble NCPs were extracted by dialysis against 250 mM citrate,pH 3.1 for 24 hours at 6° C. The residue was pelleted by centrifugation(10,000×g at 6° C. for 30 min), defatted with 1:1 (v/v)chloroform:methanol for 24 hr at 23° C., collected by filtration anddried at 22° C. The material was resuspended in 4 M GuHCl, dialyzedagainst 4 M GuHCl, 0.2% (v/v) Triton X-100, 100 mM Tris-HCl, pH 7.2 for24 hr at 6° C., then dialyzed against water, and centrifuged at 10,000×gfor 30 min at 6° C. The pellet was lyophilized and subsequentlyseparated by hydroxyapatite chromatorgraphy.

Chromatography was conducted using a BioLogic chromatography workstationwith a CHT-10 ceramic hydroxyapatite column (BioRad, Hercules, Calif.).Bovine BMP/NCP was solublized in 6 M urea, 10 mM sodium phosphate, pH7.4. The sample was loaded onto the hydroxyapatite column and theunbound fraction was collected. Bound proteins were eluted withincreasing concentration of sodium phosphate to 300 mM over a lineargradient of five column volumes. Five ml fractions were collected duringthe course of the run. The fraction which separated at 180 mM phosphatewas separated further by SDS-PAGE electrophoresis. A band correspondingto a M_(r) of 18.5 was excised and submitted for sequence analysis bymatrix assisted laser-desorption ionization/time of flight massspectroscopy (MALDI/TOF MS).

Results: Sequence Identification and Analysis: The fraction of bBMP/NCPwhich eluted from hydroxyapatite at 180 mM phosphate was separated bySDS-PAGE electrophoresis and the material with a M_(r) of 18.5 kD wassubmitted for MALDI/TOF MS analysis. The major protein component of thismaterial was determined to be a fragment of SPP-24 on the basis of sixpeptides with sequences identical to regions of that protein. (Hu, etal., Isolation and molecular cloning of a novel bone phosphoproteinrelated in sequence to the cystatin family of thiol protease inhibitors.J. Biol. Chem. 270:431-436, 1995.) The sequences of these peptides areshown in Table 1.

TABLE 1 Identification of the 18.5 kD protein by MALDI/TOF mass spectroscopy  and peptide fingerprinting. SEQExpected Observed Peptide ID Mass ^(a) Mass ^(a) Sequence NO: 1526.5741526.53 ESEADPATCDFQR * 29 1411.600 1411.71 VNSQSLSPYLER 30 1291.4061291.41 SRGEPLYEPSR 31 1249.409 1249.48 NSYLLGLTPDR 32 1158.363 1158.27GYHVPVAVCR * 33 * modified cystein; a = peptide masses are expressed as[M + H⁺]

Analysis of this sequence with the SWISS-PROT data base revealed thecystatin-like domain which had been previously described, but no othersequence similarities of relevance to bone metabolism. (Hu, et al.)However, it is known from other work that other cystatin-like proteinsinteract with proteins having a role in bone metabolism. Specially,members of the cystatin family have TGF-β and BMP-2 binding propertiesbased on similarities to the TGF-β receptor. (Brown, et al., Friends andrelations of the cystatin superfamily—new members and their evolution.:Protein Sci. 6:5-12, 1997; Demetriou, et al., Fetuin/α2-HS glycoproteinis a transforming growth factor-β type II receptor mimic and cytokineantagonist. J. Biol. Chem. 271:12755-12761, 1996.) However, fetuinantagonizes BMP activity. (Hu, et al.) Therefore, a manual comparisonwas made of the cystatin-like region of SPP-24 and the cystatin-likedomain of fetuin.

FIG. 1B is a partial amino acid sequence of the bovine SSP-24, the BMP-2homology region, and the TGF-β receptor II homology domain. Underlinedamino acids have been confirmed to be present by mass spectroscopy.(GenBank Accession Number U08018; Hu, et al.)

Two regions of interest were identified in the cystatin-like region ofSPP-24. One region had some sequence similarity to BMP-2, whereas theother region had sequence similarity to the TGF-β receptor II homologydomain of fetuin. That part of the sequence of SPP-24 which containsthese two regions is shown in FIG. 1B.

Comparisons of the two regions of interest to human BMP-2 and humanTGF-β receptor II are shown in FIGS. 2 and 3. FIG. 2 is an amino acidsequence alignment of human BMP-2 and the BMP-2 homology region inbovine SPP-24. FIG. 3 is an amino acid sequence alignment of bovinefetuin and human TGF-β receptor II (top) and of human TGF-β receptor IIand the TGF-β receptor II homology domain of bovine SPP-24(corresponding to BBP)(bottom). Alignment of the SPP-24, fetuin, humanBMP-2, and human TGF-β receptor II sequences was accomplished using theT-Coffee program. (Notredame, et al, T-Coffee: A novel method formultiple sequence alignments. J. Molecular Biol. 302:205-217, 2000.)Synthetic peptides corresponding to these two regions were obtained andsubjected to chemical and in vivo analysis as described below.

EXAMPLE 2 In Vivo Activity of Bbp

Methods: The osteogenic activity of material was tested using maleSwiss-Weber mice aged 8 to 10 weeks were used (Taconic Farms,Germantown, N.Y.). Prior to the assay, the BBP was solublized andlyophilized into 2 mg of atelocollagen. The dried material was placed ina #5 gelatin capsule and sterilized by exposure to chloroform vapor. Toconduct the assay, mice were anesthetized using 1% isoflurane deliveredin oxygen at 2 l/min through a small animal anesthesia machine(VetEquip, Pleasanton, Calif.) Animals were affixed to a surgery boardand the fur over the hindquarters shaved. The skin was cleaned with 70%ethanol and a midline incision made over the spine adjacent to thehindquarters. Blunt dissection with scissors was used to expose thequadriceps muscle on one side. A small pouch was made in the muscleusing the point of scissors and the #5 capsule containing the testmaterial was inserted into the pouch. The skin was then closed withthree 11 mm Michel surgical clips and the animal returned to its cagefor monitoring.

After 21 days the animals were killed and the hindquarter removed.Radiological examination of the specimens was accomplished using a smallparts X-Ray cabinet (Faxitron, Wheeling, Ill.). For quantization of boneformation, bone area and the bone mineral content (BMC) of an area ofinterest encompassing the site of ectopic bone formation was determinedusing a PIXImus2 small animal densitometer (GE Lunar, Madison, Wis.).Specimens were then placed in buffered formalin and submitted forroutine processing for histological examination.

Various amounts of rhBMP-2 and BBP were combined and prepared forimplantation. All possible combinations of the following amounts wereused in pilot studies, rhBMP-2: 0 μg, 0.05 μg, 0.5 μg, 5 μg, and 50 μg;BBP: 0 μg, 50 μg, and μg 500 mg. Samples of 5 μg of rhBMP-2 were used inmore extensive subsequent studies because that amount consistentlyproduced an amount of ectopic bone that was neither too large nor toosmall for reliable analysis.

Results: BBP was tested alone and in combination with rhBMP-2.

FIG. 4 is a radiogram of mouse hindquarters 21 days after implantationof 500 μg of BBP in atelocollagen (top) or atelocollagen alone (bottom).When implanted alone with carrier, BBP induced calcification.

FIG. 5 is a histological section of mouse muscle 21 days afterimplantation of 500 μg of BBP in atelocollagen. Note the dystrophiccalcification primarily associated with intramuscular adipose tissue. (H& E stain. Original magnification 100×.)

When 500 μg of BBP with sequence similarity to the TGF-β receptor II wasimplanted with 5 μg of rhBMP-2 the amount of ectopic bone formed, asmeasured by densitometry, was consistently greater than the amount ofbone formed in animals into which identical amounts of the rhBMP-2 alonewere implanted.

FIG. 6 are radiograms of mouse hindquarters 21 days after implantationof 5 μg of rhBMP-2 (left) or 5 μg of rhBMP-2 plus 500 mg of BBP (right).Note the increased opacity associated with the samples containing bothrhBMP-2 and BBP.

Furthermore, implants that contained both the peptide and rhBMP-2produced detectable cartilage and bone earlier than implants of BMP-2alone.

FIG. 7 are radiograms of mouse hindquarters 9 (above) and 12 (below)days after implantation of 5 μg of rhBMP-2 (left) or 5 μg of rhBMP-2plus 500 mg of BBP (right). Note the appearance of calcification in thesample from the day 9 sample containing both rhBMP-2 and BBP but not thesample containing BMP-2 alone.

FIGS. 8A-B are histological sections of mouse hindquarters 9 days afterimplantation of 5 μg of rhBMP-2 alone (8A) or 5 μg of rhBMP-2 plus 500μg of BBP (8B). Note the abundant cartilage in the BMP+BBP specimenwhereas the BMP alone specimen shows the earlier stages of inflammationand mesodermal cell proliferation.

TABLE 2 Densitometric quantitation of ectopic bone formation withvarious amounts of BBP implanted with 5 μg of rhBMP-2. Mean, SE (n). BBP(μg) 0 50 500 Bone Area 0.089 ± 0.0336  0.159 ± 0.0606  0.226 ± 0.0270(cm²) (12)*  (8) (12)*  Bone 0.00189 ± 0.00084 0.00388 ± 0.0017 0.00528± 0.00068 Mineral (12)** (8) (12)** Content (g) *p = 0.0044; **p =0.0049

EXAMPLE 3 Surface Plasmon Resonance to Determine the Interaction ofBMP-2 and the Synthetic Peptide

Methods: The binding interaction between rhBMP-2 and BBP wascharacterized using surface plasmon resonance employing a Biacom Xinstrument (Biacore, Piscataway, N.J.). Buffers and chips for theprocedure were obtained from Biacore. RhBMP-2 was dialyzed into 10 mMsodium acetate, pH 5.5 at a concentration of 1 mg/ml. This material wasthen attached to a CM-5 sensor chip using reagents and proceduressupplied by the manufacturer. Running buffer was 10 mM HEPES, pH 7.4,150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20. The peptide was dissolvedin running buffer at concentrations ranging from 1×10⁻⁵ to 1×10⁻⁴ M.Flow rates from 5 to 50 μl/min and injection volumes of 20 to 100 μlwere employed. The regeneration solution was 10 μM glycine-HCl, pH 2.0.

Results: Results of the surface plasmon resonance studies to determinethe interaction between rhBMP-2 and BBP are shown in FIG. 9.

FIG. 9 is a surface plasmon resonance sensogram for the interaction ofrhBMP-2 (affixed to the chip) and cyclized BBP at concentrations rangingfrom 1×10⁻⁵ M 1×10⁻⁴ M. The estimated dissociation constant (K_(D)) forthe interaction was 3×10⁻⁵ M. When the BBP was decyclized by priorreduction with β-mercaptoethanol, no significant binding occurred.

EXAMPLE 4 Residence Time Study: BBP and rhBMP-2

Methods: Labeled rhBMP-2 was mixed with BBP or vehicle and applied tocollagen sponges. The sponges were implanted into muscle pouches inrodents. At specified times (1, 3 and 7 days), the implants were removedand the amount of BMP remaining determined. Four animals were used ineach group.

Results: BBP increased retention of rhBMP-2 by a factor of about two.FIG. 10 is a bar graph depicting the percentage of rhBMP-2 retentionover 1, 3 and 7 days in the presence or absence of BBP.

Discussion: Increasing the retention of BMP at an implant site mayimprove the effectiveness of the BMP, and also reduce the amountrequired for the same therapeutic result.

While the specification describes particular embodiments of the presentinvention, those of ordinary skill can devise variations of the presentinvention without departing from the inventive concept.

EXAMPLE 5 In Vivo Activity of human BBP

Methods: The methods of Example 5 were utilized to test the activity ofhBBP in eight mice in the hindquarter ectopic bone formation assaymethod using 5 μg rhBMP-2 alone (control) or 5 μg rhBMP-2 plus 0.05 mghuman BBP (hBBP). After 4 weeks, the animals were killed and thehindquarter removed. X-ray and DEXA analysis were conducted.

Results: hBBP was tested in combination with rhBMP-2.

When implanted, hBBP with BMP resulted in a greater amount ofcalcification induction than BMP alone.

TABLE 3 Densitometric quantitation of ectopic bone formation withvarious amounts of BBP implanted with 5 μg of rhBMP-2. Mean, SE (n).Group Mean BMC content (g) rhBMP-2 (5 μg) 0.00775 hBBP (0.05 mg) +rhBMP-2 (5 μg) 0.01125

EXAMPLE 6

The Effect of BBP on Spinal Fusion as an Alternative of Adjunct to BMPin a Relevant Animal Model

The rat posterior spinal fusion model was used to investigate the effectof BBP on fusion.

Methods: Three groups of 6 Lewis rats each underwent posterolateralintertransverse process spinal fusion at vertebrae L4-L5 with theapplication of collagen sponges containing: BBP (500 μg) (Group1); BBP(500 μg) plus low-dose rhBMP-2 (1 μg) (Group 2); collagen plus low-doserhBMP-2 (1 μg) (Group 3). Results were compared to historical controlsof decortication only (0% fusion) and high-dose rhBMP-2 (3 μg) (100%fusion). All rats underwent post-operative radiography at 2, 4, 6, and 8weeks. Manual spinal palpation was performed at 8 weeks, the time ofsacrifice. Bone formation was assessed in histological sections stainedwith H&E.

Results: At week 8, the fusion rates were: 14% in Group 1 (BBP only);80% in Group 2 (BBP plus low-dose BMP); and 40% in Group 3 (collagenplus low-dose BMP). This compares to 0% for historical negative controlsat 100% for historical positive controls.

TABLE 4 Results of fusion rates by treatment group Groups 2. BBP (500μg) + 3: collagen plus low- 1: BBP collagen plus low-dose dose rhBMP-2(500 μg) rhBMP-2 (1 μg) (1 μg) Fusion Rates 14% 80% 20%

Discussion: Higher fusion rates in Group 2 (BBP plus low-dose BMP) asopposed to Group 3 (collagen plus low-dose BMP) suggest that BBP has anadjunctive effect on BMP that could permit the use of lower doses of BMPto achieve spinal fusion. Further studies with larger numbers ofsubjects are required to confirm these promising results.

EXAMPLE 7 The Adjunctive Effect of a Binding Peptide on BMP in an AnimalSpinal Fusion Model

To investigate the effects of BBP on spinal fusion as an alternative oran adjunct to BMP in a relevant animal model.

Methods: Five groups of 50 Lewis rats each underwent posterolateralintertransverse process spinal fusion at vertebrae L4-L5 with implanted:BBP (500 μg) (Group 1); BBP (1000 μg) (Group 2), collagen plus low doseBMP-2 (1 μg) (Group 3), BBP (500 μg) plus low dose BMP (1 μg) (Group 4)and BBP (1000 μg) plus low dose BMP (1 μg) (Group 5). Control groupsincluded a negative group (decortication) and positive control group(collagen plus 3 μg BMP).

All rats underwent post-operative radiography at 2, 4, 6, and 8 weeks.Manual spinal palpation was performed at 8 weeks, after the time ofsacrifice. Bone formation was assessed in histological sections stainedwith H&E.

Results: At week 8, manual palpation of revealed 0% fusion with groups 1and 2 (low d high dose BBP only), 40% fusion in group 3 and 90% fusionin groups 5 (BBP 1000 μg_low dose BMO). The difference between group 3and 5 was close to statistical significance (p=0.056). Historicalcontrol groups as negative group (only decortation) resulted with a 0%fusion while the fusion rate with the positive control group (full doseBMP) was 100% in all our previous studies. The rate of radiographicfusion was significantly higher in group 5 than in group 3 at the 4thand 6th week (p<0.05) while it was nearly significant at the 8th week(p=0.056). Histological analysis revealed a more mature and thicker bonemass in group 4 and 5 when compared to group 3.

Discussion: Higher fusion rates with BBP plus low-dose BMP than collagenplus low-dose BMP may indicate an adjunctive effect of BBP on BMPpossibly enabling the use of lower doses of BMP for spinal fusion in thefuture.

EXAMPLE 8 The Adjunctive Effect of a Binding Peptide on BMP in an AnimalSpinal Fusion Model

A prospective 8 week interventional trial employing a rat model ofspinal fusion to test the effect on bone morphogenetic protein bindingpeptide (BBP) on rhBMP-2 induced bone healing. The objective of thisstudy was to determine if the addition of BBP to the collagen spongesused as a carrier for rhBMP-2 reduces the amount of rhBMP-2 required toachieve a satisfactory clinical outcome. BBP was effective as an adjunctto increase the effect of BMP-2 and enabled decreasing the dose of theprotein necessary to provide fusion.

Methods: Posterolateral intertransverse process spinal fusion at L4-L5was performed in five treatment groups of Lewis rats each, and collagensponges (5×5×13 mm) were implanted into the surgical sites.

Each sponge was placed in a sterile microfuge tube and either asuspension of BBP (500 μg or 1000 μg) in water or sterile water alonewas applied. The sponges were allowed to air dry in a tissue culturehood overnight and then placed at −70° C. for an hour and lyophilizedovernight. The tubes (with small holes in the top to allow forlyophilization) were placed in autoclave pouches and sealed. Thematerials were then sterilized by exposure to chloroform vapor for atleast 4 hours Immediately prior to surgery, the pouches were opened andthe sponges were removed from the tubes and placed in a second tubecontaining the designed amount of rhBMP-2 or water. The sponges wereable to completely absorb the solution.

The posterolateral intertransverse process spinal fusion at L4-L5 in therat is a well established procedure in our laboratory 13-17. Briefly,the animal was anesthetized with isofluane and the surgical site wasshaved and prepped with Betadyne and alcohol. A 3 cm longitudinalmidline incision was made through the skin and subcutaneous tissue overL4-L5 down to the lumbodorsal fascia. Then, two separate 2 cmlongitudinal paramedial incisions were made in the erector spinaemuscles on both sides of L4-L5. The transverse processes of L4-L5 wereexposed, cleaned of soft tissue, and decorticated with a high-speedburr. The site was irrigated with saline and the therapeutic testmaterial was placed. The lumbodorsal fascia was closed with 4-0 Prolene(Ethicon, Somerville, N.J.). The skin was closed with 4-0 Prolene andmeticulous post-operative care was provided.

Two doses of BBP (500 μg, and 1000 μg) were tested with or without “lowdose” (1 μg) rhBMP-2 and the results were compared to the low dose (1μg) rhBMP-2. These were compared to control groups. Fusion was evaluatedby radiology, histology and manual palpation tests. Animals weresacrificed 8 weeks after surgery.

The results of the surgeries were assessed radiologically,histologically, and by manual palpation. Posteroanterior radiographswere obtained using a small parts X-ray cabinet (Faxitron Corp.,Wheeling, Ill.) on each animal at 2, 4, 6 and 8 weeks. Radiograms wereevaluated by three independent observers employing the followingstandardized scale: 0: no fusion; 1: incomplete fusion with boneformation present; and 2: complete fusion. The scores from the threeobserves were added and a total of 5 or 6 was regarded as “fused”.Manual palpation of the spine was performed at the time of sacrifice at8 weeks. To simulate the gold standard for determining fusion in humans,exploration and manual palpation of the fusion mass were performed afterdeath. The level fused was manually palpated with Adson forceps andcompared with the adjacent nonfused levels. Each specimen was graded asfused or non-fused by three independent observers. The spine wasdesignated as “not fused” if any of the three observers graded the spineas not fused. After manual palpation, the specimens were decalcifiedusing standard 10% decalcifying solution HCl (Cal-Ex, Fisher Scientific,Fairlawn, N.J.), washed with running tap water, then transferred to 75%ethanol. Sagittal sections were cut carefully at the level of thetransverse process. The specimens were imbedded in paraffin and sectionsof each specimen obtained. These sections were stained with hematoxylinand eosin. They were evaluated by an independent observer as fused andnot fused.

The proportions of subjects in each group judged to be “fused” werecompared in sequential two group comparisons with Fisher's exact testusing SPSS software. A p score of less than or equal to 0.05 was set forstatistical significance.

Results: Radiology revealed significant earlier fusion with 1000 μgBBP+1 μg BMP-2. combination when compared to low dose BMP-2 (1 μg) only(p<0.05). Manual palpation and histology at 8th week revealed higherrate of fusion with the same combination with a nearly significantdifference (p=0.057).

TABLE 5 Results of radiological examination Week 2nd 4th 6th 8th (%) (%)(%) (%) Group 1: low dose BBP only (500 μg) 0 0 0 0 Group 2: high doseBBP only (1000 μg) 0 0 0 0 Group 3: low dose BMP-2 only (1 μg 0 20 30 40rhBMP-2) Group 4: low dose BBP + low dose BMP-2 0 20 80 80 Group 5: highdose BBP + low dose BMP-2 40 80 90 90 Group 6: decortication only — — —0 Group 7: high dose BMP-2 only (10 μg — — — 100 rhBMP-2)

TABLE 6 Results of manual palpation and histology at 8th week. HistologyManual (%) Palpation (%) Group 1: low dose BBP only (500 μg) 0 0 Group2: high dose BBP only (1000 μg) 0 0 Group 3: low dose BMP-2 only (1 μg40 40 rhBMP-2) Group 4: low dose BBP + low dose BMP-2 80 80 Group 5:high dose BBP + low dose BMP-2 90 90 Group 6: decortication only — —Group 7: high dose BMP-2 only — 100

The results of the radiological examinations are shown in Table 5 whichpresents the proportion of subjects in each group that had a score of 5or 6 and was, therefore, judged to be “fused” for each of the four timepoints (2, 4, 6 and 8 weeks). As can be seen none of the animals in lowand high dose BBP groups developed fusion at any time points. Low doseBBP+low dose BMP-2 group had a higher fusion rate than BMP-2 low doseonly group at the 6th and 8th weeks and this difference was significant(p<0.05) for the 6th week while not significant at the 8th week(p=0.170). On the other hand BBP high dose+BMP-2 low dose group had asignificantly higher fusion rate at the 4, 6 and 8 week time points whencompared to BMP-2 low dose only group (p<0.05 for each time point)(FIGS. 15 and 16).

Table 6 shows the proportions of subjects in each group judged to be“fused” by three independent clinical evaluators. The criticalcomparison was between BMP-2 low dose group versus BBP low dose+BMP-2low dose and BBP high dose+BMP-2 low dose groups. The fusion rateassessed by manual palpation was higher in both combination groups whencompared to BMP-2 low dose group (FIGS. 15 and 16). However, thisdifference was not significant while it tended towards significance forthe comparison of BBP high dose+BMP-2 low dose group with BMP-2 low dosegroup (p=0.057). The thickness of the fusion mass tended to be thickerand the maturity of the bone tended to be more mature in the specimensof the fused combination groups when compared to the specimens of thefused low dose BMP-2 group (FIGS. 17 and 18). FIG. 17 is a histologicalsection of rat spinal region 8 weeks after treatment of a combination ofBBP and rhBMP-2 showing thick fusion mass between L4 and L5 transverseprocess with cortical-matured bone. (H & E stain. Original magnification8.4×). FIG. 18 is a histological section of rat spinal region 8 weeksafter treatment with low dose rhBMP-2 (1 μg) showing immature bonebridging the cortical matured bridging bony parts. (H & E stain.Original magnification 8.4×).

Table 6 also shows the proportion of subjects in each group judged to befused by an independent histologist. The results confirmed the resultsof both the manual palpation and radiology at the 8th week demonstratingthe similar fusion rates. The thickness of the fusion mass tended to bethicker and the maturity of the bone tended to be more mature in thespecimens of the fused combination groups when compared to the specimensof the fused low dose BMP-2 group (FIGS. 17 and 18).

Specific growth factor binding agents, such as BBP, can be compoundedinto carriers used in fusion procedures to decrease the dosage of BMPand possibly decrease the side effects which are most likelydose-related. This may also decrease costs and improve clinicaloutcomes.

The radiographic results demonstrated significantly higher and earlierfusion rates with high dose BBP+low dose BMP-2 combination when comparedto the same amount of the protein in its single use. The histology andthe manual palpation tests confirmed this higher rate of fusion at the8th week with a nearly significant difference. It is important to notethat this model of spinal fusion is difficult to fuse and requires amaterial with significant osteoinductive ability to induce a solidarthrodesis. Likewise, use of either 500 μg or 1000 μg BBP alone did notprovide fusion in the spinal model, while there was ectopic dystrophiccalcification formation with 500 μg BBP implantation in the muscle pouchmodel. In the histological examination, we have observed a more maturebone fusion mass with the combination groups when compared with theBMP-2 only group (FIGS. 17 and 18).

We claim:
 1. A method of increasing the rate bone formation invertebrate tissue, comprising applying to a tissue a peptide having thesequence of SEQ ID NO: 11, or a fragment thereof, and a dose of leastone bone growth factor, wherein the combination results in a faster rateof bone formation than treatment with the same dose of the at least onebone growth factor alone.
 2. The method of claim 1 wherein the bonegrowth factor selected from a group of molecules or compounds which bindwith the peptide to increase the retention rate of the growth factor atthe tissue.
 3. The method of claim 1 wherein the bone growth factor is aTGF-β family member.
 4. The method of claim 1 wherein the bone growthfactor is selected from the group comprising: TGF-β, BMP-2, BMP-3,BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, GDF5 and, demineralized bonematrix.
 5. The method of claim 1 wherein the peptide has a sequence ofSEQ. ID NO: 1, and the bone growth factor is BMP.
 6. The method of claim1 wherein the peptide has a sequence of SEQ. ID NO: 12, and the bonegrowth factor is hBMP.
 7. The method of claim 1 wherein bone formationis measured as ostegenesis.
 8. The method of claim 1 wherein boneformation is measured by calcification.
 9. A method of increasing therate of vertebrate bone fusion, comprising applying to a tissue apeptide having the sequence of SEQ ID NO: 11, or a fragment thereof, anda dose of least one bone growth factor, wherein the combination resultsin a faster rate of bone fusion than treatment with the same dose of theat least one bone growth factor alone.
 10. The method of claim 9 whereinthe bone growth factor selected from a group of molecules or compoundswhich bind with the peptide to increase the retention rate of the growthfactor at the tissue.
 11. The method of claim 9 wherein the bone growthfactor is a TGF-β family member.
 12. The method of claim 9 wherein thebone growth factor is selected from the group comprising: TGF-β, BMP-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, GDF5 and, demineralizedbone matrix.
 13. The method of claim 9 wherein the peptide has asequence of SEQ. ID NO: 1, and the bone growth factor is BMP.
 14. Themethod of claim 9 wherein the peptide has a sequence of SEQ. ID NO: 12,and the bone growth factor is hBMP.
 15. A method of inducing the rate ofvertebrate bone fusion, comprising applying to a tissue a peptide havingthe sequence of SEQ ID NO: 11, or a fragment thereof, and a dose ofleast one bone growth factor, wherein the combination results in afaster rate of bone formation than treatment with the same dose of theat least one bone growth factor alone.
 16. The method of claim 15wherein the bone growth factor selected from a group of molecules orcompounds which bind with the peptide to increase the retention rate ofthe growth factor at the tissue.
 17. The method of claim 15 wherein thebone growth factor is a TGF-β family member.
 18. The method of claim 15wherein the bone growth factor is selected from the group comprising:TGF-β, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, GDF5 and,demineralized bone matrix.
 19. The method of claim 15 wherein thepeptide has a sequence of SEQ. ID NO: 1, and the bone growth factor isBMP.
 20. The method of claim 15 wherein the peptide has a sequence ofSEQ. ID NO: 12, and the bone growth factor is hBMP.
 21. A method ofinducing the rate of vertebrate bone fusion, comprising applying to atissue a peptide having the sequence of SEQ ID NO: 11, or a fragmentthereof, and a dose of least one bone growth factor, wherein thecombination results in a faster rate of bone formation than treatmentwith the same dose of the at least one bone growth factor alone.
 22. Themethod of claim 21 wherein the bone growth factor selected from a groupof molecules or compounds which bind with the peptide to increase theretention rate of the growth factor at the tissue.
 23. The method ofclaim 21 wherein the bone growth factor is a TGF-β family member. 24.The method of claim 21 wherein the bone growth factor is selected fromthe group comprising: TGF-β, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7,BMP-8, BMP-9, GDF5 and, demineralized bone matrix.
 25. The method ofclaim 21 wherein the peptide has a sequence of SEQ. ID NO: 1, and thebone growth factor is BMP.
 26. The method of claim 21 wherein thepeptide has a sequence of SEQ. ID NO: 12, and the bone growth factor ishBMP.
 27. A method of inducing the rate of membranous bone fusion,comprising applying to a tissue proximate to membranous bone, a peptidehaving the sequence of SEQ ID NO: 11, or a fragment thereof, and a doseof least one bone growth factor, wherein the combination results in afaster rate of bone formation than treatment with the same dose of theat least one bone growth factor alone.
 28. The method of claim 27wherein the bone growth factor selected from a group of molecules orcompounds which bind with the peptide to increase the retention rate ofthe growth factor at the tissue.
 29. The method of claim 27 wherein thebone growth factor is a TGF-β family member.
 30. The method of claim 27wherein the bone growth factor is selected from the group comprising:TGF-β, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, GDF5 and,demineralized bone matrix.
 31. The method of claim 27 wherein thepeptide has a sequence of SEQ. ID NO: 1, and the bone growth factor isBMP.
 32. The method of claim 27 wherein the peptide has a sequence ofSEQ. ID NO: 12, and the bone growth factor is hBMP.
 33. An isolatedpolypeptide for increasing bone formation, comprising SEQ ID NO: 11, andwherein the polypeptide increases the residency time of at least onebone growth factor.
 34. The method of claim 33 wherein the peptide has asequence of SEQ ID NO:
 1. 35. The method of claim 33 wherein the peptidehas a sequence of SEQ ID NO:
 12. 36. An isolated nucleic acid forencoding a polypeptide for increasing bone formation, comprising SEQ IDNO: 11, and where in the polypeptide increases the residency time of atleast one bone grown factor.
 37. The method of claim 36 wherein thenucleic acid has a sequence of SEQ ID NO:
 2. 38. The method of claim 36wherein the nucleic acid has a sequence of SEQ ID NO:
 21. 39. Acomposition for use in bone formation comprising: a peptide having thesequence of SEQ ID NO: 11, or a fragment thereof, and a dose at leastone growth factor where in the composition results in faster rate ofbone formation than the same dose of the at least one growth factoralone.
 40. The composition of claim 39 where in the peptide has asequence of SEQ ID NO:
 1. 41. The composition of claim 39 where in thepeptide has a sequence of SEQ ID NO:
 12. 42. The composition of claim 39wherein the growth factor is selected from the group comprising: TGF-β,BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, GDF5 and,demineralized bone matrix.